N-Rich is a twin-column chromatography process that enriches target compounds relative to other components in a mixture, thereby facilitating their isolation and characterization. This study demonstrates the performance of N-Rich for isolation of Angiotensin II peptide impurities compared with standard analytical and preparative chromatography approaches.
Journal of Chromatography A 1667 (2022) 462894 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Tutorial Article Enrichment and purification of peptide impurities using twin-column continuous chromatography Richard Weldon, Thomas Müller-Späth#,∗ ChromaCon AG, Technoparkstr 1, Zurich CH-8005, Switzerland a r t i c l e i n f o Article history: Received 30 December 2021 Revised February 2022 Accepted February 2022 Available online 16 February 2022 Keywords: Peptide purification Continuous chromatography N-Rich Impurity isolation Impurity profiling a b s t r a c t N-Rich is a twin-column chromatography process that enriches target compounds relative to other components in a mixture, thereby facilitating their isolation and characterization This study demonstrates the performance of N-Rich for isolation of Angiotensin II peptide impurities compared with standard analytical and preparative chromatography approaches Peptides have diverse chemical properties and are produced using a wide range of methods, resulting in products with complex impurity profiles The characterization of impurities for clinical development is essential but obtaining high purity samples in sufficient quantities is often a difficult task when using standard chromatographic techniques In contrast, by using cyclic continuous chromatography with UV-based process control, N-Rich enables automatic oncolumn accumulation of target impurities while other compounds in the mixture are depleted This has multiple advantages compared to standard techniques Firstly, at the end of the cyclic accumulation phase the highly enriched target is eluted in one step with high purity and concentration This means fewer fractions for analysis are generated and up-concentration steps are reduced Secondly, the purification of target impurities using semi-preparative scale chromatography becomes viable, even if initial resolution is poor compared to analytical HPLC This allows for very significant increases in productivity for purification of difficult to isolate impurities This study demonstrates two N-Rich strategies: Example 1: Purification of μg quantities of multiple Angiotensin II impurities with a >9-fold increase in productivity compared to analytical HPLC Example 2: Specific isolation of mg of a critical impurity at 88% purity 79-fold increase in productivity and a 69-fold reduction in solvent consumption compared to analytical HPLC © 2022 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Introduction Therapeutic proteins, peptides and oligonucleotides are produced by chemical or biological synthesis After synthesis, in addition to the active pharmaceutical ingredient, all drug products include product-related impurities, process-related impurities, and degradation products According to ICH guidelines [1–3], impurities comprising >0.1% of the total drug product require identification and characterization as part of risk assessment regarding their impact on patient safety and product efficacy Based on this assessment, a product specification is defined with strict limits for every impurity to guarantee product safety and efficacy For complex molecules such as peptides, oligonucleotides and protein biologics, the number of impurities with abundance >0.1% can be ∗ # Corresponding author E-mail address: thomas.mueller-spaeth@chromacon.com (T Müller-Späth) https://www.chromacon.com considerable A bottleneck to completing impurity characterization is the lack of availability of highly purified material Milligram amounts of such material are often required for structural confirmation by NMR or X-ray crystallography, and biological studies for toxicity, immunogenicity and pharmacokinetics can require even more [4–6] 1.1 Standard isolation of impurities vs N-Rich To complete the task of impurity isolation, column chromatography is often the method of choice However, even if a protocol is well optimized, standard chromatographic methods have technical limitations leading to a tradeoff between productivity and purity [7] For example, standard HPLC techniques use stationary phases with very small particle diameters combined with a low sample load to maximize resolution and purity, but at the expense of productivity This approach is suitable for tentative identification of impurities using very sensitive methods such as LC-MS but is https://doi.org/10.1016/j.chroma.2022.462894 0021-9673/© 2022 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) R Weldon and T Müller-Späth Journal of Chromatography A 1667 (2022) 462894 Fig The N-Rich process is carried out as shown in the schematic The black diagonal line indicates the steepness of the gradient (%B) Design: With the aid of software, the boundaries for target recycling (blue, green) are set using a batch chromatogram as template Four N-Rich methods are generated: Startup - Column is loaded with a full batch load Enrichment - target compounds (blue, green) undergo “n” cycles of enrichment while undesirable compounds (red) are discarded as new feed is applied Each cycle of enrichment consisted of switches Each “switch” is composed of four phases of elution, P1, P2, P3 & P4 as shown in Fig table A full description of the enrichment process is detailed in the main text Depletion – A single switch is carried out without loading new feed resulting in depletion of non-target compounds before the final elution step Elution + Fractionation – Finally, enriched target impurities are eluted with fractionation To maximize resolution the final method uses a shallow gradient, low flow rate, and double the bed height compared to the enrichment phase Table Materials – Analytical, semi-preparative batch and N-Rich chromatography Method Material Description Analytical HPLC Column Mobile phase A Mobile phase B Feed composition Feed purity Column Mobile phase A Mobile phase B Feed composition Feed purity Column Mobile phase A Mobile phase B In-line dilution buffer Feed composition Feed purity ˚ 1.7 μm, 2.1 mm x 50 mm) ACQUITY UPLC Oligonucleotide BEH C18 Column (130 A, vol% acetonitrile (Merck, 1.00030) / 0.1 vol% TFA (VWR UN2699) in water 90 vol% acetonitrile / 0.1 vol% TFA in water mg/mL in Mobile phase A 92% YMC Triart Prep C18-S (150 × 4.6 mm ID, S-10 μm, 12 nm) vol% acetonitrile / 0.1 vol% TFA in water 50 vol% acetonitrile / 0.1 vol% TFA in water 2.1 mg/mL in Mobile phase A 92% 2x YMC Triart Prep C18-S (150 × 4.6 mm ID, S-10 μm, 12 nm) vol% acetonitrile / 0.1 vol% TFA in water 50 vol% acetonitrile / 0.1 vol% TFA in water vol% acetonitrile / 0.1 vol% TFA in water 2.1 mg/mL in Mobile phase A 92% Semi-Preparative Batch N-Rich less suited for production of μg or mg quantities required for more detailed studies To obtain larger quantities of target impurities for characterization the conventional solution is to pool impuritycontaining fractions from multiple analytical HPLC runs until sufficient material has been accumulated However, difficult to separate or low abundant impurities can require hundreds or thousands of analytical purifications over many weeks or months to generate enough material Alternatively, the impurity may need to be chemically synthesized and purified [4] By contrast, semi-preparative chromatography using stationary phases with larger particle diameter and higher loading capacities is ideal if an impurity is well resolved from neighboring compounds However, semi-preparative chromatography often has insufficient resolution of critical target impurities resulting in low purity fractions which leaves analytical HPLC as the only viable standard chromatographic option N-Rich is a twin-column chromatographic process with the potential to improve upon standard methods by facilitating the recycling and enrichment of target compounds on-column, and in a continuous manner The process can be generally applied for the isolation of impurities from complex mixtures using standard gradient purification methods as a starting point For example, N-Rich was successfully applied for monoclonal antibody (mAb) charge variant isolation [8,9] using ion-exchange chromatography showing a 30-fold decrease in the time required to obtain 10 mg compared to analytical HPLC [9] Moreover, the purity obtainable with N-Rich was higher than the purity by analytical HPLC (95% vs 85% purity) R Weldon and T Müller-Späth Journal of Chromatography A 1667 (2022) 462894 Table Semi-preparative batch run parameters Semi-Prep Batch Method Column dimensions Equilibration Load Gradient start Gradient end Strip Re-equilibration Equilibration Loading Wash after load Gradient Strip Re-equilibration Equilibration Load Wash after load Gradient duration Strip Re-equilibration Run time Batch [mL] [%B] [g/L] [%B] [%B] [%B] [%B] [cm/h] [cm/h] [cm/h] [cm/h] [cm/h] [cm/h] [CV] [CV] [CV] [CV] [CV] [CV] [h:min] 0.525 0.25 7:20 Batch Batch (4.6 mm ID x 30 cm) 15 1.05 2.1 15 70 100 15 500 300 300 100 500 500 0.5 22 7:21 7:24 Batch N-Rich Example – Design Batch N-Rich Example – Design Batch 2.5 (4.6 mm ID x 15 cm) 15 8.4 0.525 8.4 15 85 100 15 500 300 300 300 500 500 0.25 25 10 8:30 0:44 1:04 Table N-Rich run parameters Procedure N-Rich Example Method Startup Column dimensions Equilibration Feed load Wash after load Gradient start Gradient end In-line dilution Strip Re-equilibration Equilibration Loading Wash after load Gradient flow rate (initial) Gradient flow rate (W-rec & P-collect) Gradient flow rate (S-Rec) Strip Re-equilibration In-line dilution flowrate (W-rec) In-line dilution flowrate (S-Rec) AutoPeak threshold Equilibration Load Wash after load Gradient duration Strip Re-equilibration Cycles done Feed processed Buffer consumption Run time (Method) Run time (Procedure) [mL] – [g/L] [%B] [%B] [%B] – – – [cm/h] [cm/h] [cm/h] [cm/h] [cm/h] [cm/h] [cm/h] [cm/h] factor factor mAU [CV] [CV] [CV] [CV] [CV] [CV] – [mg] [L] [h:min] [h:min] IL Dil Buffer 0.525 15 – – – – – 600 200 200 – – – – – – – – 0.25 – – – 1.32 0:11 Enrichment N-Rich Example Depletion Elution Startup Enrichment Depletion × 2.5 (4.6 mm ID x 15 cm) – – – IL Dil Buffer – – 0.412 – – 8.4 5.25 – – – – 15 – – 15 15 15 – 15 15 85 85 70 – 59 59 IL Dil Buffer IL Dil Buffer – – IL Dil Buffer IL Dil Buffer Mob pH-B Mob pH-B Mob pH-B – Mob pH-B Mob pH-B IL Dil Buffer IL Dil Buffer IL Dil Buffer – IL Dil Buffer IL Dil Buffer – – – 600 – – 200 – – 300 300 – – – – 300 – – 300 300 100 – 300 300 253 253 – – 130 130 199 199 – – 216 216 500 – 600 600 600 600 600 600 500 – 600 600 2.57 2.57 – – – – 3.55 3.55 – – 3 390 390 – – – – – – – – – 0.196 – – 2.5 – – – – – – 8 22 – 6.32 6.32 2 – 2 3 – 3 18 1 36.96 0 21 210 3.03 1.08 19:21 0:37 7:06 0:26 7:11 0:27 27:15 16:04 Elution – – – 15 55 – Mob pH-B IL Dil Buffer – – – 100 – – 500 500 – – – – – – 25 8:00 1.2 The N-Rich process principle So given the high utility of N-Rich for antibody charge variant isolation, this study aims to evaluate the potential of N-Rich to obtain peptide impurities in a more productive way In this study, reversephase chromatography was used to exploit displacement effects [10,11] which are less pronounced in ion-exchange chromatography, but are very useful to obtain target compounds with high purity In contrast to other twin-column chromatographic processes such as Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) [12] or CaptureSMB [13], the N-Rich process is not designed to reach a cyclic steady state with multiple product elutions for obtaining product with uniform concentration and purity Instead, in N-Rich, the target compounds are accumulated from cycle to cycle A step-by-step depiction of the N-Rich process principle is outlined in Fig The N-Rich design procedure is described below in section 4.2 N-Rich is composed of four steps run as an uninterrupted procedure The enrichment principle is detailed in the table in Fig The table shows the activities of column and column during a single N-Rich switch: Initial state – At the very beginning of an N-Rich run before the enrichment phase begins, column is pre-loaded in a startup step while column is clean This pre-load allows non-target compounds to reach a cyclical steady state quicky which facilitates their removal using UV-based dynamic process control During the R Weldon and T Müller-Späth Journal of Chromatography A 1667 (2022) 462894 Fig Chromatograms of crude Angiotensin II (92% purity) comparing analytical HPLC and semi-preparative chromatography ⱡ = “easy” to isolate impurity; (∗ ) = “difficult” to isolate impurity A - Analytical reversed phase (C18) HPLC B – “Zoom in” of A C - Semi-preparative (C18) chromatography comparing different loads D – “Zoom in” of C E - A fraction analysis of an Angiotensin II preparative batch chromatogram (8.4 g/L load) Critical target impurity (∗ ) is obtainable at a purity of 8% cyclical enrichment phase, column is always fully loaded at the completion of the prior switch, while column requires cleaning and regeneration to remove strongly adsorbing non-target impurities • • • P1 (columns in parallel) - column undergoes elution of weakly adsorbing, non-target compounds to waste; column simultaneously undergoes regeneration ready for the loading steps in P2, P3 and P4 P2 (columns interconnected for target recycling) – column undergoes elution of weakly adsorbing target impurities (W), recycled with in-line dilution directly to column The in-line dilution ensures re-adsorption of the target impurities on column P3 (columns in parallel) – column undergoes elution of nontarget compounds (P) to waste; column is simultaneously loaded with new feed This step allows enrichment of the tar- • get compounds relative to non-target compounds in the mixture To maintain a switch-to-switch steady state, the quantity of new feed applied is in equilibrium with non-target products removed The load required to achieve this is calculated with the aid of software, based on the batch chromatogram and fraction analysis P4 - (columns interconnected for target recycling) – column undergoes elution of strongly adsorbing target impurities (S), recycled with in-line dilution directly to column Again, the in-line dilution ensures re-adsorption of the target impurities on column At the conclusion of switch 1, both columns are immediately ready for switch Column is now fully loaded and column requires cleaning and regeneration P1 – P4 are repeated as described above, but with column positions interchanged (Fig table - yellow arrows) By alternating the position of column and column R Weldon and T Müller-Späth Journal of Chromatography A 1667 (2022) 462894 Fig A batch chromatogram is the starting point of N-Rich design A – Example uses a low load (0.5 g/L) and a resulting chromatogram with resolution of all impurities except one species (∗ ) that co-elutes with the main compound Two enrichment windows were applied covering the early eluting (blue) and late eluting (green) target impurities B - N-Rich example was designed using a high load (8.4 g/L) Whilst overall resolution of impurities was reduced, the high load is advantageous for the specific enrichment of target impurity (∗ ) that co-elutes with the tail of the main conpound peak (green) The higher load increased the area of the main compound peak (red) that contains no target impurity (∗ ) thus allowing a higher load during the enrichment phase and higher productivity switch-to-switch, N-Rich can operate continuously resulting in the incremental enrichment of the target impurities inside the system A single cycle of N-Rich is defined as switches that includes elution from each column and the enrichment phase is continued for “n” cycles, until sufficient feed is processed Following completion of the enrichment step a depletion step is carried out This consists of a single switch without loading new feed so that only the enriched target compounds are transferred to the next column before starting the final elution step This greatly reduces the amount of non-target compounds present in the final elution step Finally, the enriched target material is eluted with a very shallow gradient over two columns in-series and the target material is collected with a fine fractionation Thus, a high resolution of the enriched compounds is obtained, and the pure target material is collected by pooling suffi- ciently pure fractions only once Another benefit of the enrichment process is that the impurity concentration in the collected fractions is much higher than compared to standard chromatographic methods As a model for impurity isolation, we used crude Angiotensin II as starting material Angiotensin II is a human hormone which functions as a vasoconstrictor and can be used as a medication to treat hypotension The therapeutic version is a synthetic 8-amino acid peptide marketed under the brand name Giapreza® Since the peptide is relatively short, it is produced by solid phase peptide synthesis (SPPS) In general, SPPS is a highly efficient process (97% coupling reaction efficiency), but with many coupling steps and just as many deprotections steps, it is common to generate many impurities which differ from the target peptide in a minor way, for example by missing a single intermediary amino acid This study R Weldon and T Müller-Späth Journal of Chromatography A 1667 (2022) 462894 Fig N-Rich Example Absorbance at 220 nm; column – red line, column – blue line; Regions of enrichment - blue & green shading, region of depletion - red shading A – Enrichment step – “Zoom out” - 18 Cycles enrichment phase Inset - “Zoom in” of column enrichment profile B – Depletion step – switch with no new feed C Final elution step - columns in series (2x bed height, 2x CV gradient, 0.5x flow rate compared to enrichment phase) 2.2 Batch and N-Rich chromatography evaluates the twin-column N-Rich process compared to single column chromatography for generating large quantities of low abundant impurities from crude Angiotensin II obtained by SPPS Semi-preparative batch methods were carried out for comparison with N-Rich as well as to serve as the process design template for N-Rich Batch and continuous chromatography were carried out using the Contichrom CUBE 30, a lab-scale twin column system for continuous chromatography (ChromaCon AG, A YMC Company) UV absorbance at A220 nm was recorded by external UV detectors (2x Knauer AZURA UVD2.1S, ADA00 + 0.5 mm flow cells, A4069) located directly after each column outlet (UV1@220 nm and UV2@220 nm respectively) and connected to the Contichrom CUBE system Table details the column characteristics, buffer composition and feed composition used for semi-preparative batch chromatography and N-Rich runs Table gives an overview of the method parameters used for semi-preparative batch runs and N-Rich design runs (Batch 1, & = Fig 2C and 2D Batch = Fig 2E, N-Rich Design Batch = Fig 3A and 3B) Table gives Methods 2.1 Analytics Analytical HPLC chromatography was carried out using an Agilent 1290 series system set at 60 °C Table shows the column, solvents and feed used in the method The gradient parameters and flow rate are detailed in Supplemental Table Samples from the Angiotensin II feed, the batch fractions & N-Rich fractions were prepared for HPLC analysis by diluting 1:1 with water Samples were analyzed at a wavelength of A220 nm Fraction concentrations were also calculated using the area under the curve (AUC) measured from analytical HPLC R Weldon and T Müller-Späth Journal of Chromatography A 1667 (2022) 462894 Fig N-Rich Example – Analytical Evaluation A – 11 enriched fractions were analyzed by HPLC, and chromatograms were overlayed with the feed profile B – “Zoom in” of the feed profile shows the level of impurities in the feed relative to the main Angiotensin II peak The failure to enrich the early eluting impurity (∗ ) that co-elutes with the main compound was notable This impurity was targeted separately in N-Rich example The table at the bottom of Fig indicates the total mass obtained per 1.5 mL fraction an overview of the method parameters used for two N-Rich example runs shown in Fig and Fig from a semi-preparative batch method (Fig 2E) Note that, when comparing analytical and semi-preparative scale, selectivity reversal of impurity (∗ ) was seen As loading was increased from 0.5 g/L to 1.0 and 2.0 g/L resin, it was also observed that additional early eluting impurities (ⱡ, Fig 2D) were no longer resolved from the main compound peak as the Langmuirian peak profile broadened to the left This further limits the utility of batch methods with a higher load than 0.5 g/L In conclusion, compared to analytical HPLC, semi-preparative batch chromatography is limited due to a lack of resolution of early eluting target impurities (∗ , ⱡ) For later eluting impurities, loads up to g/L did not severely impact the separation performance (Fig 2C and 2D, 250 – 350 min) For these late eluting impurities semipreparative batch chromatography could be a good alternative to analytical HPLC Given the limitations of these standard chromatographic approaches, N-Rich was tested as an alternative process for impurity production Results and discussion 3.1 Limitations of standard chromatography for impurity isolation A high resolution analytical HPLC chromatogram of crude Angiotensin II purified using an analytical reversed phase C18 column is shown in Fig 2A and 2B The purity of the crude material is 92% and there are multiple low-level impurities present on either side of the main compound peak Whilst analytical HPLC provides excellent resolution of impurities and main compound, the very low loading capacity of the analytical stationary phase severely limits the productivity of HPLC for impurity production Purification of μg quantities of these impurities would require pooling of hundreds of chromatographic runs, taking weeks of run time As an alternative to analytical HPLC, semi-preparative chromatographic methods give worse resolution due to the larger stationary phase particle diameters (10 μm compared to 1.7 μm) However, if sufficient resolution of impurities is achievable, this is a preferable option for impurity isolation as semi-preparative stationary phases are cheaper, have a higher loading capacity and can run at high flow rates with lower backpressure Thus, the semi-preparative scale allows for far higher productivity, provided that the target purity can be achieved To explore the viability of semi-preparative chromatography for Angiotensin II impurity isolation a high resolution batch method was carried out To maximize resolution, the method utilized a very shallow gradient, a low flow rate and longer columns than analytical HPLC (Fig 2C and 2D) While a generally good resolution was achievable with a load of 0.5 g/L (the lower limit at which impurities remained detectable), compared to analytical HPLC, a critical target impurity (∗ ) completely co-eluted with the main compound peak (Fig 2B vs 2D) This impurity, which was well resolved in analytical HPLC, had a resulting purity of only 8% in fractions 3.2 N-Rich design procedure Two complementary N-Rich designs were tested N-Rich was firstly used to purify a broad spectrum of Angiotensin II peptide impurities simultaneously and then used in a more targeted way to accumulate one difficult to isolate impurity (∗ ) A pre-requisite to design N-Rich is to generate a single column batch “design” chromatogram using the Contichrom CUBE system An N-Rich design method serves as a template for the cyclical enrichment step and is typically optimized for productivity rather than very high resolution, since high resolution is only advantageous in the final elution and fractionation step and not required in the impurity accumulation step of the process After running the design method, the resulting chromatogram (Fig 3A and 3B) was imported into the N-Rich design software (N-Rich wizard, a module within the Contichrom CUBE operating software) Using the wizard, the gradient (15%B to 85%B) of the chromatogram was then R Weldon and T Müller-Späth Journal of Chromatography A 1667 (2022) 462894 Fig N-Rich Example Absorbance at 220 nm; Column – red line, Column – blue line; Regions of enrichment - green shading, Region of depletion - red shading A Cycle enrichment phase B - Final elution through columns in series (2x bed height, 2x CV gradient, 0.5x flow rate compared to enrichment phase) divided into phases, as described in Fig 1., using five adjustable section borders (Fig 3A and 3B, t1-t5) Once section borders for impurity recycling and non-target compound removal were configured, the wizard automatically determined the necessary load per switch and in-line dilution factors (Table 3) In addition, NRich example required a UV-threshold-based control set point (AutoPeak) to trigger main compound removal to waste This was necessary to stabilize performance by compensating for small deviations in the retention time that occur during a long, multi-cycle enrichment phase Upon finalization of the method parameters, the N-Rich wizard automatically created four methods as described in Fig and outlined in Table (Startup, Enrichment, Depletion & Final Elution) trum of impurities in one procedure (Fig 3A) A total of 18 enrichment cycles were run to process 38.2 mg of feed, after which the concentration of the impurities on column was sufficiently high to carry out the depletion and final elution steps Fig 4.A shows the results of the enrichment phase where cyclical overlays were used to visualize the accumulation of the impurities The load applied per switch was in equilibrium with the main compound removed (red shaded area) so as impurities accumulate, the main peak is always in steady state The UV-based process control successfully compensated for changes in the retention time Fig 4.B shows the final switch without feeding (depletion) before the final elution step was carried out (Fig 4C) Total processing time was ≈27 h A clear increase in resolution was seen between the depletion step and the final elution step due to the use of a high-resolution method Also evident in the final elution step is the depletion of the main compound peak relative to the target impurities and this results in very little of the Angiotensin II main compound present 3.3 Example 1: N-Rich for broad isolation of Angiotensin II impurities N-Rich example used a small load of 0.4 g/L resin per switch to maximize resolution with the aim of accumulating a broad spec8 R Weldon and T Müller-Späth Journal of Chromatography A 1667 (2022) 462894 Fig N-Rich Example – evaluation of enriched fractions by analytical HPLC A – Quantification of product vs critical impurity (∗ ) using analytical HPLC, overlayed with final N-Rich elution chromatogram The % purity of the target is reported next to the data labels for each fraction B – Analytical HPLC overlay of input feed vs fraction with highest enrichment of critical impurity (∗ ) (90% pure) The table shows the % purity, mass recovered, and % yield of the highest purity fraction vs a pool of high purity fractions in any of the impurity fractions The fractions were collected and analyzed for purity using analytical HPLC Fig shows an analytical overlay of 11 fractions containing enriched impurities compared to the original feed Most regions of the chromatogram were enriched except for two impurities (4 & 5(∗ )) which were co-eluting with the main compound and sent to the waste Impurity 5(∗ ) was targeted separately in NRich example below Depending on the starting abundance of a particular impurity in the feed, between 13 μg and 58 μg was obtained after N-Rich fractionation The purity of the weakly adsorbing impurities was between 80% - 94%, whereas strongly adsorbing impurities were overlapping with their neighbors resulting in lower purities between 48% and 75% In theory, purity of these strongly adsorbing impurities could be further im- proved by using a shallower gradient in this region of the chromatogram and collecting smaller fractions in the final elution step By comparison, a well optimized analytical HPLC method used to accomplish the same task (assuming 50-μg load per run, a 30 run time, a high purity of 85–95%), would require >500 chromatography runs and 10 days of continuous running time (>9x longer than N-Rich) Finally, a high-resolution semi-preparative batch method would give similar purity to N-Rich, but with a load of 0.5 g/L per run, a total of 15 runs taking 4.6 days would be required to complete the task (4x longer than N-Rich), and the pool concentration would be 15x lower, meaning up-concentration (with potential sample loss) would be an essential step before subsequent analysis R Weldon and T Müller-Späth Journal of Chromatography A 1667 (2022) 462894 Fig Process performance comparison of optimized analytical HPLC, semi-preparative batch chromatography, and N-Rich chromatography for the purification of mg of critical impurity (∗ ), in terms of purity, processing time and solvent consumption In summary, N-Rich example shows that a broad enrichment strategy facilitates fast purification of μg quantities of multiple peptide impurities simultaneously and produced at higher concentration than analytical or semi-preparative batch chromatography This is ideal for generating samples for tentative characterization using, for example, MS analysis For generating larger quantities with high purity, the following N-Rich example demonstrates a second approach (56% recovery) Possibly, additional cycles would further improve purity, but that remains to be tested HPLC, semi-preparative batch, and N-Rich chromatography were compared in terms of purity, processing time and solvent consumption (Fig 8) to obtain mg impurity (∗ ) that is present at 0.8% content in the starting material The analytical HPLC method used in the comparison represents a hypothetical, optimized analytical HPLC allowing a loading of 50 μg per HPLC run The actual HPLC method used in the experimental part of this study was not well adapted for the task of impurity production because a load of